Lassa Fever Virus Glycoprotein Mediates Lamp1-And Low Ph-Dependent Cell-Cell Fusion Through A Stalk-Pore Mechanism

Lassa fever virus (LASV) GP glycoprotein-mediated entry into cells proceeds through multiple distinct steps. Initial binding to the surface α-dystroglycan allows virus uptake and transport to late endosomes/endolysosomes. Here acidic pH mediates virus dissociation from α-dystroglycan and binding to the LAMP1 receptor that is thought to trigger LASV fusion. The difficulties associated with mechanistic studies of virus-endosome fusion prompted us to examine LASV GP-mediated fusion using a cell-cell fusion model. We show that cells expressing LASV GP fuse to mammalian cells upon exposure to low pH and that this fusion is promoted by ectopic expression of LAMP1 and, even more significantly, by the LAMP1 mutant that resides in the plasma membrane. These findings suggest endogenously expressed LAMP1 is present on the cell surface. Using this new cell-cell fusion model, we show that LASV GP does not require priming by a receptor to undergo irreversible acid-mediated refolding. Additional mechanistic experiments showed that, similar to other viral glycoproteins, LASV GP-mediated fusion proceeded through a hemifusion intermediate, which can be arrested by exposure to low pH in the cold, and that this intermediate can be converted to full fusion by chlorpromazine treatment. We further demonstrate that GP-induced hemifusion requires the formation of a highly curved lipid intermediate (stalk) disfavored by incorporation of a positive-curvature lipid lyso-phosphatidylcholine into the plasma membrane. Real-time measurements of transfer of fluorescent cytoplasmic markers revealed that LASV GP-mediated fusion pores did not enlarge as readily as those formed by other viral glycoproteins. The less efficient dilation of these fusion pores might be due to the difference in lipid/protein composition of late endosomes and the plasma membrane. Ongoing experiments are examining the role of lipids in LASV-mediated cell-cell fusion.